8530
J. Org. Chem. 1998, 63, 8530-8535
Isolation, Structural Assignment, and Synthesis of N-(2-Methyl-3-oxodecanoyl)-2-pyrroline, a New Natural Product from Penicillium brevicompactum with in Vivo Anti-Juvenile Hormone Activity Pilar Moya,† A Ä ngel Cantı´n,† Maria-Angeles Castillo,‡ Jaime Primo,† Miguel A. Miranda,† and Eduardo Primo-Yu´fera*,† Instituto de Tecnologı´a Quı´mica UPV-CSIC and Departamento de Quı´mica, Universidad Polite´ cnica de Valencia, 46022 Valencia, Spain Received December 15, 1997
A new natural product with in vivo anti-juvenile hormone (JH) activity, N-(2-methyl-3-oxodecanoyl)2-pyrroline (2), has been isolated from Penicillium brevicompactum Dierckx. Its structure has been tentatively assigned based on spectral data and unambiguously confirmed by alternative syntheses. Compound 2 has been prepared by means of a sequence of reactions beginning with acylation of Meldrum’s acid by octanoyl chloride. The subsequent steps have been aminolysis of the resultant intermediate with pyrrolidine, alkylation of the active position with iodomethane, introduction of a methoxy group in the pyrrolidine ring by anodic oxidation, and final elimination of methanol through adsorption on SiO2 and heating. The natural product 2 and its bicyclic isomer 2-heptyl3-methyl-4-oxo-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b]-1,3-oxazine (3) are also obtained. This reveals that 2 can be biogenetically related to the recently discovered brevioxime (1). Compound 2 and the synthetic precursors of 2 have shown important biological activities as insecticides (against Oncopeltus fasciatus Dallas) and fungicides. The natural product induces precocious metamorphosis in the target insect (70% precocious adults at 10 µg/nymph). In view of the above results, these products could be useful as lead molecules for the synthesis of analogues with enhanced biological activities. Introduction One of the most important and challenging aspects of pesticide research is the urgent need to develop new and effective methods of controlling plagues; these methods should cause no harm to human health and the environment, and they must be accepted as safe by the general public.1 Natural products, with their tremendous structural diversity, are an important source of new alternatives. Many natural products showing fungicidal, bactericidal, or insecticidal activities are isolated every year. If their properties allow and if sufficient quantities can be obtained from natural sources or by synthesis, such compounds may be used as agricultural chemicals. Alternatively, they may constitute useful starting points as lead molecules for the synthesis of analogues with improved biological and physical properties.2 In recent times, investigations by several research groups have shown that one of the most important sources of bioactive compounds is fungus. The secondary metabolites of fungal origin exhibit a wide range of potentially useful biological activities.3,4 †
Instituto de Technologı´a Quı´mica. Departamento de Biotecnologia de la Universidad Polite´cnica de Valencia, 46022 Valencia, Spain. * Correspondence: Instituto de Tecnologı´a Quı´mica UPV-CSIC, Universidad Polite´cnica de Valencia, Avenida de los Naranjos s/n, Apartado 22012, 46022 Valencia, Spain. Fax: 34-6-3877809. Phone: 34-6-3877807. E-mail:
[email protected]. (1) Richardson, M. L., Ed. Chemistry, Agriculture and Environment; The Royal Society of Chemistry: Cambridge, 1991. (2) Pillmoor, J. B.; Wright, K.; Terry, A. S. Pestic. Sci. 1993, 39, 131140. (3) Omura, S. J. Ind. Microbiol. 1992, 10, 135-156. (4) Porter, N.; Fox, F. M. Pestic. Sci. 1993, 39, 161-168. ‡
Penicillium is one of the genera, together with Aspergillium and Fusarium, which produce metabolites known to be toxic to insects.5 Particularly, the fungus Penicillium brevicompactum Dierckx has been described as one of the most prolific producers of secondary metabolites. These include mycophenolic acid and related compounds,6 the Raistrick phenols,7-9 the pebrolides10 or the N-benzoyl derivatives of phenylalanine, phenylalaninol, and its ester, asperphenamate.11 In addition, the fungus also produces brevigellina,12 several piperazine-2,5-dione derivatives, a drimane diterpenoid,13 the brevianamides,14,15 and compactin.16 The latter is a reported hypocholesterolaemic agent that was shown to be a reversible, competitive inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMG-CoA reductase). Previous work on the effects of this product on (5) Wright, V. F.; Vesonder, R. F.; Ceigler, A. In Microbial and Viral Pesticides; Kurstak, E., Ed.; Marcel Dekker, Inc.: New York, 1982; pp 559-587. (6) Birkinshaw, J. F.; Raistrick, H.; Ross, D. J. Biochem. J. 1952, 50, 630-634. (7) Oxford, A. E.; Raistrick, H. Biochem. J. 1932, 27, 1902-1906. (8) Oxford, A. E.; Raistrick, H. Biochem. J. 1933, 27, 634-652. (9) Godin, P. Biochim. Biophys. Acta 1955, 11, 114-118. (10) McCorkindale, N. J.; Calzadilla, C. H.; Hutchinson, S. A.; Kitson, D. H.; Ferguson, G.; Campbell, I. M. Tetrahedron 1981, 37, 649-653. (11) Doerfler, D. L.; Bird, B. A.; Campbell, I. M. Phytochemistry 1981, 20, 2303-2304. (12) McCorkindale, N. J.; Baxter, R. L. Tetrahedron 1981, 37, 17951801. (13) Ayer, W. A.; Altena, I. V.; Browne, L. M. Phytochemistry 1990, 29 (5), 1661-1665. (14) Birch, A. J.; Wright, J. J. Tetrahedron 1970, 26, 2329-2344. (15) Birch, A. J.; Russell, F. A. Tetrahedron 1972, 28, 2999-3002. (16) Brown, A. G.; Smale, T. C.; King, T. J.; Kasenkamp, R.; Thompson, R. H. J. Chem. Soc., Perkin Trans. 1 1976, 1165-1170.
10.1021/jo972267v CCC: $15.00 © 1998 American Chemical Society Published on Web 10/29/1998
N-(2-Methyl-3-oxodecanoyl)-2-pyrroline
insects has shown that it is able to produce a potent in vitro juvenile hormone (JH) biosynthesis inhibition, with IC50 of the order of 10-7-10-9 M in lepidoptera17,18 and dictioptera.19,20 However, as a general rule, the morphological effects of compactin are scarce. Recently we have reported the isolation and identification of brevioxime (1), a new metabolite from P. brevicompactum, which exhibits a very high activity as a JH biosynthesis inhibitor.21 Its chemical structure contains an unusual heterobicyclic skeleton and an oxime functionality. In this paper we report the isolation, identification, and alternative synthesis of a new natural product (2) from P. brevicompactum, with a high in vivo anti-JH activity.
Although at first sight the structures of 1 and 2 seem unrelated, chemical studies have shown that 2 can be converted into the bicyclic isomer 3 upon acid catalysis; and because basic skeletons of 1 and 3 are the same, it appears that the new natural product 2 can be biogenetically related to brevioxime. Results and Discussion A systematic screening was performed with 118 strains of Penicillium isolated from fungal contamination of cereals. The most promising results were obtained with a strain of P. brevicompactum. A summary of the procedure followed to isolate the active compound is illustrated in Scheme 1. The dichloromethane extract obtained from the culture medium exhibited the highest entomotoxicity and anti-JH activity to Oncopeltus fasciatus (20% mortality and 40% precocious adults at 10 µg/cm2, following the method of Bowers et al.22). In (17) Monger, D. J.; Lim, W. A.; Kerdy, F. J.; Law, J. H. Biochem. Biophys. Res. Commun. 1982, 105, 1374-1380. (18) Hiruma, K.; Yagi, S.; Endo, A. Appl. Entomol. Zool. 1983, 18, 111-115. (19) Edwards, J. P.; Price, N. R. Insect Biochem. Mol. Biol. 1983, 13, 185. (20) Belle´s, X.; Camps, F.; Casas, J.; Lloria, J.; Messeguer, A.; Piulachis, M. D.; Sanchez, F. J. Pestic. Biochem. Physiol. 1988, 32, 1-10. (21) Moya, P.; Castillo, M.; Primo-Yu´fera, E.; Couillaud, F.; Martı´nez-Ma´n˜ez, R.; Garcera´, M. D.; Miranda, M. A.; Primo, J.; Martı´nezPardo, R. J. Org. Chem. 1997, 62, 8544-8545. (22) Bowers, W. S.; Ohta, T.; Cleere, J. S.; Marsella, P. A. Science 1976, 193, 542-547.
J. Org. Chem., Vol. 63, No. 23, 1998 8531 Scheme 1. Isolation and Purification of the Anti-JH Compound 2 from P. brevicompactum
addition, the extract showed an important fungicidal activity against Colletotrichum gloesporoides, Alternaria tenuis, Fusarium culmorum, and Trichoderma viride at 500 µg/mL. The entomotoxicity and anti-JH activity bioassay served as a guide for silica gel column separation, which led to a fraction (F8) with remarkable in vivo anti-JH activity. Preparative HPLC of this fraction allowed isolation of the active pure compound Pb/8A. Its structure was tentatively assigned, by means of combined spectral data, as N-(2-methyl-3-oxodecanoyl)-2-pyrroline (2). To confirm the structure and to prepare higher amounts of compound for further biological assays, we designed an alternative synthesis based on the approach outlined in Scheme 2. If successful, slight modifications of this approach could allow synthesis of a number of related intermediates and derivatives with enhanced biological activities or both. The synthetic scheme involves the use of commercial starting materials such as pyrrolidine and octanoyl chloride. The first step was acylation23,24 of Meldrum’s acid25,26 with octanoyl chloride. This treatment gave an intermediate, which was submitted to aminolysis without previous purification, by reaction with pyrrolidine in refluxing benzene.27 This led to the β-ketoamide 4 in 61% overall yield from octanoyl chloride. The next step was introduction of a methyl group28,29 between both carbonyls. Thus, after using NaH to (23) Oikawa, Y.; Sugano, K.; Yonemitsu, O. J. Org. Chem. 1978, 43, 2087-2088. (24) Oikawa, Y.; Yoshioka, T.; Sugano, K.; Yonemitsu, O. Org. Synth. 1984, 62, 198. (25) Meldrum, A. N. J. Chem. Soc. 1908, 93, 598-601. (26) Davidson, D.; Bernhardt, S. A. J. Am. Chem. Soc. 1948, 70, 3426. (27) Pak, C. S.; Yang, H. C.; Choi, E. B. Synthesis 1992, 1213-1214. (28) Benetti, S.; Romagnoli, R. Chem. Rev. 1995, 95, 1065-1114. (29) Abad, A.; Agullo´, C.; Arno´, M.; Cantı´n, A.; Cun˜at, A. C.; Meseguer, B.; Zaragoza´, R. J. J. Chem. Soc., Perkin Trans. 1 1997, 1837-1843.
8532 J. Org. Chem., Vol. 63, No. 23, 1998 Scheme 2.
Moya et al. Synthesis of the Natural Ketoamide 2
generate the carbanion, methylation was achieved by treatment with iodomethane. The desired alkylated β-ketoamide 5 was obtained in 79% yield. To achieve the pyrrolidine to pyrroline conversion of 5, a methoxy group was introduced at C2 by means of anodic oxidation, following the method previously described by Shono.30-35 A methanolic solution of β-ketoamide 5 was subjected to a constant electric current of 20 mA, in the presence of tetrabutylammonium ptoluenesulfonate as a supporting electrolyte, until 3.7 F/mol had passed through the solution. In this way, the methoxylated β-ketoamide (6) was obtained in 45% yield, and 50% of the starting material was recovered. The two diasteromers of 6 (a and b) were resolved by column chromatography. Both of them were present in solution as a mixture of the two possible amide conformations, which gave separate signals in NMR. Finally, elimination of methanol was carried out by adsorption of 6 on SiO2 and subsequent heating to 150160 °C.31,36-40 Under these conditions, a 1:1 mixture of the desired pyrroline 2 and the isomeric bicyclic product 3 was obtained. (30) Shono, T. Tetrahedron Lett. 1984, 40, 811-850. (31) Shono, T.; Matsumura, Y.; Tsubata, K.; Sugihara, Y.; Yamane, S.; Kanazawa, T.; Aoki, T. J. Am. Chem. Soc. 1982, 104, 6697-6703. (32) Shono, T.; Matsumura, Y.; Tsubata, K.; Sugihara, Y. Tetrahedron Lett. 1982, 23, 1201-1204. (33) Shono, T.; Hamaguchi, H.; Matsumura, Y. J. Am. Chem. Soc. 1975, 97, 4262-4268. (34) Shono, T.; Matsumura, Y.; Tsubata, K. J. Am. Chem. Soc. 1981, 103, 1172-1176. (35) Shono, T.; Matsumura, Y.; Tsubata, K. Tetrahedron Lett. 1981, 22, 2411-2412. (36) Slomczynska, U.; Chalmers, D. K.; Cornille, F.; Smythe, M. L.; Beusen, D. D.; Moeller, K. D.; Marshall, G. R. J. Org. Chem. 1996, 61, 1198-1204. (37) Cornille, F.; Fobian, Y. M.; Slomczynska, U.; Beusen, D. D.; Marshall, G. R.; Moeller, K. D. Tetrahedron Lett. 1994, 35, 6889-6992. (38) Cornille, F.; Slomczynska, U.; Smythe, M. L.; Beusen, D. D.; Marshall, G. R.; Moeller, K. D. J. Am. Chem. Soc. 1995, 117, 909917. (39) Moeller, K. D.; Rutledge L. D. J. Org. Chem. 1992, 57, 63606363. (40) Moeller, K. D.; Hanau, C. E.; Avignon, A. Tetrahedron Lett. 1994, 35, 825-828.
Table 1.
Entomotoxic and In Vivo Anti-JH Activity against O. Fasciatus
productsa
dose (µg/cm2)
toxicity %b
anti-JH activity %c
2 4
10 10.0 7.5 5.0 10.0 7.5 5.0 2.5 1.0
20.0 ( 4.5 91.1 ( 3.9 56.7 ( 4.7 6.7 ( 0.0 100 100 82.2 ( 2.2 24.4 ( 4.6 13.3 ( 0.0
71.4 ( 5.4e ndd nd nd
5
nd nd nd
a Products without activity are not reflected in the table. b Each value means the average (n ) 3) and deviation standard of percentage of mortality, with respect to control. Results were obtained by the contact method. c Percentage of precocious adults with respect to surviving nymphs in the entomotoxic test. d nd: not detected. e Anti-JH activity has been detected by topical application, on newly moulted fourth-instar nymphs, at 10 µg/ nymph.
Insecticidal, anti-JH, and antimicrobial activities of the natural product 2, as well as for the bicyclic isomer 3 and the synthetic intermediates 4-6, have been evaluated. Table 1 contains the relevant data for the compounds showing activity against the milkweed bug O. fasciatus. The natural product (2) exhibited an important antagonistic JH activity that induced precocious metamorphosis. The effects of this product on treated nymphs were of the same type as those described for the precocenes.22,41 In addition, 2 has been shown to be a true anti-JH agent according to Staal,42 because its coadministration with a juvenoid (methoprene) is able to reverse the action. Experiments are under way to determine the mechanism of action of this compound. On the other hand, two intermediates in the synthesis of 2, compounds 4 and 5, have shown a strong knockdown toxicity to O. fasciatus (41) Bowers, W. S. Discovery of Insect Antiallatotropins in The Juvenile Hormones; Gilbert, L. I., Ed. Plenum Press: New York, London, 1976. (42) Staal, G. B. Annu. Rev. Entomol. 1986, 31, 391-429.
N-(2-Methyl-3-oxodecanoyl)-2-pyrroline
J. Org. Chem., Vol. 63, No. 23, 1998 8533
Table 2.
Fungicidal Activities of Products 2-6 percentage of radial mycelial growth inhibitiona % (mean ( SD)b
target phytopathogens
2
3
4
5
6a
6b
Fusarium culmorum Fusarium oxysporium ssp. gladioli Fusarium oxysporium ssp. niveum Geotrichum candidum Colletotrichum gloesporoides Colletotrichum coccodes Trichothecium roseum Alternaria tenuis Rosellinia necatrix Verticillium dahliae Trichoderma viride Penicillium italicum Aspergillus parasiticus
0 0
